CN109731015B - Immunity enhancer based on hirsutella sinensis and preparation method - Google Patents
Immunity enhancer based on hirsutella sinensis and preparation method Download PDFInfo
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Abstract
The invention relates to an immunopotentiator based on hirsutella sinensis and a preparation method thereof. An immunopotentiator based on hirsutella sinensis is white dry powder, mainly contains saccharide, amino acid and its derivatives and nucleoside compounds, has effects of enhancing immunity, and can improve human immunity, and has wide market. The method optimizes the culture of hirsutella sinensis by adding metabolic inducers and the like, so that the immunocompetent substances in the culture are increased; the optimal extraction process of the immune active substances in the hirsutella sinensis fermentation product is obtained by optimizing the extraction and chromatographic separation preparation processes. The immune active substance is mainly obtained from hirsutella sinensis culture solution, and hirsutella sinensis mycelia serving as a byproduct can also replace wild cordyceps sinensis to process related health-care products, so that the immune active substance can be obtained by killing two birds with one stone. The invention is produced by indoor fermentation, can be produced in an industrialized way all the year round, is not influenced by the harsh environment of plateau low temperature and the like of the wild cordyceps sinensis, and does not damage the ecological environment.
Description
Technical Field
The invention belongs to the technical field of biological products and preparation, and particularly relates to an immunopotentiator based on hirsutella sinensis and a preparation method thereof.
Background
The cordyceps sinensis is a famous traditional Chinese herbal medicine in China, has been applied for more than a thousand years and is famous with ginseng and pilose antler. It is a kind of worm-fungus complex formed by cordyceps fungus and parasitic insect, including the worm body portion full of hypha (sclerotium) and the fruit body portion growing from head. Modern pharmacological research shows that the cordyceps sinensis can regulate immunity, delay senility, lower blood pressure, regulate internal secretion, eliminate fatigue, prevent and treat tumor, acute or chronic hepatitis and nephritis, has antibacterial and antiviral activities and other pharmacological activities, and has important medical and health care values. Because the natural aweto has special growth environment and is easily influenced by external environment, and the natural aweto is collected artificially, the quantity of the natural aweto is continuously reduced, and the natural aweto resource is in the state of being nearly exhausted at present. In order to solve the contradiction between the exhaustion of resources and the continuous rising of demand, people strive to find a substitute for cordyceps sinensis. At present, more and more wild cordyceps products are prepared from real cordyceps sinensis hirsutella sinensis (hirsutella sinensis) (a)Hirsutella sinensis) Culture was substituted.
Research shows that the hirsutella sinensis culture also has an immunoregulation effect, but no research report about extraction and preparation of components with immunopotentiation activity in hirsutella sinensis exists at present. The immunomodulator achieves the effects of preventing and treating diseases by regulating human immunity, and is an important material basis for strengthening body resistance and eliminating evil in traditional Chinese medicines, so that intensive research and development are necessary. According to the early stage research of the invention, the content of immune active substances can be increased by improving the hirsutella sinensis culture method, and the components 3 and 7 are found to have stronger immune enhancement effect by further using an activity-guided separation method, and the extraction method of the active components is optimized to obtain the optimal extraction and preparation process.
Disclosure of Invention
The invention aims to provide an immunopotentiator based on hirsutella sinensis and a preparation method of the immunopotentiator based on hirsutella sinensis.
An immunopotentiator based on hirsutella sinensis is white dry powder; wherein the content of saccharide is 5-50%, the content of amino acid and its derivatives is 3-30%, and the content of nucleoside is 1-10%.
A preparation operation step of an immunopotentiator based on hirsutella sinensis comprises the following steps:
(1) preparation of first-order solid Strain
The separated and purified hirsutella sinensis (A), (B), (C), (Hirsutella sinensis) Inoculating slant strains in a flat solid culture medium, wherein the inoculation amount is that each slant is inoculated with 2 plates of 9 cm; culturing at 17-20 ℃ for 20-30 days to obtain a first-grade solid strain;
(2) preparation of second-stage liquid strain
Inoculating the first-stage solid strain in a liquid culture medium, wherein the inoculation amount is that 1 plate of the first-stage solid strain is inoculated in every 100mL of the liquid culture medium; shaking culture: the temperature is 17-20 ℃, and the rotating speed is 160-200 r/min; culturing for 10-20 days to obtain a secondary liquid strain;
(3) preparing three-stage liquid strain
Inoculating the secondary liquid strain into a seed tank of a fermentation system according to the volume ratio of 5-20%, wherein the liquid filling amount of a liquid culture medium of the fermentation tank is 50-70%; culturing for 5-15 days at 17-20 ℃ according to the volume ratio of 1:1 (v/v) and the pressure of 0.1MP to obtain a third-level liquid strain;
the formula of the liquid culture medium is the same as that of the liquid culture medium used by the secondary liquid strain;
(4) preparation of fermentation broth
Inoculating the third-stage liquid strain into a fermentation tank of a fermentation system according to the volume ratio of 5-10%, wherein the liquid filling amount of the fermentation tank is 50-70%; adding 100-1000 mg/L of first additive into the culture medium for 5-30 days at the temperature of 17-20 ℃ according to the ventilation volume of 1:1 (v/v) and the pressure of 0.1 MP; continuously culturing for 10-40 days, adding 1.0-100.0 mg/L second additive agent, and continuously culturing for 20-40 days; obtaining fermentation liquor;
the fermentation medium and the liquid medium have the same formula;
the additive No. one: is prepared from zinc sulfate: plant sphingosine: mannose according to a weight ratio of 0.1-0.9: 0.01-0.2: 0.1-0.5;
the second additive: hydrogen peroxide (H)2O2): potassium nitrosodisulfonate (K)2NO(SO3)2) According to the weight ratio of 0.01-0.8: 0.5-0.01;
(5) preparation of hirsutella sinensis immunopotentiator
Heating the fermentation liquor to 50.0-99.9 ℃, and keeping for 5-20 minutes; cooling to room temperature, adding ethanol with the volume of 0.1-2.0 times of that of the fermentation liquor, standing for 1-24 hours at the temperature of 0-20 ℃; centrifuging to obtain supernatant or filtering to obtain filtrate; concentrating the supernatant or the filtrate to 1/10-1/2 of the original volume, passing through a reversed phase carbon 18 chromatographic column, eluting with 0-90% ethanol, collecting the eluate after sample injection for 2-50 minutes, removing the solvent in the eluate, and recovering the ethanol to obtain the hirsutella sinensis immunopotentiator.
The preparation operation technical scheme is further defined as follows:
in the step (1), the solid medium: 200g of potato cooking juice, 20g of glucose, 10g of maltose, 10g of peptone, 10g of yeast powder and magnesium sulfate (MgSO 2)4) 1.5g, potassium dihydrogen phosphate (KH 2 PO)4) 3g of agar, 20g of agar, 1L of water was added, and the pH was adjusted to 6.5 with 1M sodium hydroxide (NaOH).
In steps (2) and (3), the liquid medium: 20g of glucose, 30g of silkworm chrysalis powder, 20g of yeast powder and magnesium sulfate (MgSO)4) 0.5g of potassium dihydrogen phosphate (KH 2 PO)4) 1g of water was added to 1L, and the pH was adjusted to 6.5 with 1M sodium hydroxide (NaOH).
In the step (3), the fermentation medium: 20g of glucose, 30g of silkworm chrysalis powder, 20g of yeast powder and magnesium sulfate (MgSO)4) 0.5g of potassium dihydrogen phosphate (KH 2 PO)4) 1g of water was added to 1L, and the pH was adjusted to 6.5 with 1M sodium hydroxide (NaOH).
In the step (4), the fermentation medium: 20g of glucose, 30g of silkworm chrysalis powder, 20g of yeast powder and magnesium sulfate (MgSO)4) 0.5g of potassium dihydrogen phosphate (KH 2 PO)4) 1g of water was added to 1L, and the pH was adjusted to 6.5 with 1M sodium hydroxide (NaOH).
And (5) concentrating the supernatant or the filtrate under the pressure of 0.0001-0.01 Mpa to 1/10-1/2 of the original volume.
In the step (5), the centrifugal separation: rotating speed of 5000-10000 r/min, and separating for 5-15 min.
In the step (5), the filtration: sieving the mixture by a sieve of 50-200 meshes.
In the step (5), the mobile phase passing through the reversed phase carbon 18 chromatographic column is 0-90% ethanol.
The invention increases the immune active substances in the culture by optimizing the culture method of hirsutella sinensis; the optimal extraction process of the immune active substances in the hirsutella sinensis is obtained by optimizing the extraction and separation preparation processes.
The beneficial technical effects of the invention are embodied in the following aspects:
1. earlier researches of the invention find that the water extract of hirsutella sinensis liquid fermentation hypha can obviously improve the transcription levels of IL-1 beta, IL-6 and TNF-alpha in mouse macrophage, and show that the hirsutella sinensis liquid fermentation hypha have the function of enhancing immunity. The improvement of the culture method can increase the content of the immune active substances in the hirsutella sinensis. The invention uses hirsutella sinensis culture to produce the immunopotentiator, and can obtain hirsutella sinensis mycelium while obtaining the immunopotentiator; the immunopotentiator based on hirsutella sinensis can improve human immunity, and hirsutella sinensis mycelia can replace wild cordyceps sinensis to process related health products, thereby achieving two purposes and having high economic benefit.
2. In the earlier stage research of the invention, the content of immune active substances can be increased by improving the culture method of hirsutella sinensis, and the components 3 and 7 (shown in the figure are omitted) are found to have stronger immune enhancement effect by further using a separation method under the guidance of activity, and the extraction method of the active components is optimized to obtain the optimal extraction and preparation process. The hirsutella sinensis immunopotentiator is produced by indoor fermentation, can be produced industrially all the year round, and is not influenced by the harsh environments such as plateau low temperature and the like of wild cordyceps sinensis.
The invention compares the difference of different cultured metabolome by a metabonomics (metabolomics) method, and finds that the zinc sulfate, the plant sphingosine, the mannose, the hydrogen peroxide, the nitroso-disulfonic acid potassium and the like can obviously improve the metabolism of the hirsutella sinensis for the first time, so that the metabolites with the function of enhancing the immunity are obviously increased. The micromolecule immunomodulator is prepared for the first time by optimizing the extraction and separation process.
3. The immunopotentiator based on hirsutella sinensis of the invention is a natural component in hirsutella sinensis, and the cordyceps sinensis and the hirsutella sinensis are widely used in health food in China, so that the safety of related products is high. The immunopotentiator is produced by artificially fermenting hirsutella sinensis, but wild cordyceps sinensis is not used, so that the ecological environment of the producing area of the wild cordyceps sinensis can be protected, and resource exhaustion and grassland degradation caused by excessive excavation are avoided. At present, the world population with low immunity is increasing, including a large number of elderly people with low immunity and people with low immunity caused by environmental pollution, high working pressure and the like, so that the related immunopotentiator has a wide market.
Detailed Description
The invention is further illustrated by the following examples.
Example 1
A preparation operation step of an immunopotentiator based on hirsutella sinensis comprises the following steps:
1) strain selection
Cordyceps collected from Qinghai [ (C.) A. Ex Fr. ] SingOphiocordyceps sinensisHirsutella sinensis isolated from stroma (Hirsutella sinensis）；
2) Culturing of bacterial strains
The culture method is liquid-solid mixed culture and comprises the following specific steps:
2.1) first order solid Strain preparation
Inoculating the separated and purified slant strains into a solid culture medium of a 9cm culture dish, wherein the culture medium comprises: 200.0 g of potato cooking juice, 20.0 g of glucose, 10.0 g of maltose, 10.0 g of peptone, 10.0 g of yeast powder and MgSO4 1.5 g、KH2PO43.0 g of agar, 20.0 g of agar, 1.0L of water, and adjusting the pH to 6.5 by using 1.0M NaOH; placing into 17 deg.C incubator, and culturing for 20 days to obtain first-stage solid strain.
) Secondary liquid seed culture
200mL of liquid culture medium is filled into a 500mL triangular flask; each 1000mL of culture medium contains glucose 20.0 g, pupa Bombycis powder 30.0 g, yeast powder 20.0 g, MgSO40.5g, KH2PO41.0 g, mixing the components, adding water to supplement the mixture to 1000mL, and adjusting the pH value to 6.5 by using 1.0M NaOH; using 1.2 Kg/cm2Autoclaving for 30 min, cooling to room temperature, inoculating 2 solid strains in culture dish to 500mL triangular flask culture medium, and culturing in constant temperature shaking incubator at 17 deg.C and 160 rpm for 10 days to obtain second-stage liquid strain.
) Three-stage liquid strain culture
400mL of liquid culture medium is filled into a 1000mL triangular flask; each 1000mL of culture medium contains 20.0 g of glucose, 30.0 g of silkworm chrysalis powder and 20g of yeast powder.0 g、MgSO4 0.5g、KH2PO41.0 g, mixing the components, adding water to supplement the mixture to 1000mL, and adjusting the pH value to 6.5 by using 1.0M NaOH; using 1.2 Kg/cm2Autoclaving for 30 min, cooling to room temperature, inoculating the second-stage liquid seeds into the cooled liquid culture medium at 5%, and culturing at 17 deg.C and 160 rpm for 10 days to obtain third-stage liquid strain.
) Quaternary fermentation and metabolic regulation
Inoculating the third-stage liquid strain into a fermentation tank according to the volume ratio of 5%, wherein the liquid filling amount of the fermentation tank is 50%, the ventilation amount is 1:1 (v/v), the pressure is 0.1MP, and the first additive with the concentration of 100 mg/L is added after the fermentation tank is cultured for 5 days at the temperature of 17 ℃; continuously culturing for 10 days, and adding 1.0 mg/L second additive to obtain fermented product.
The formula of the first additive comprises zinc sulfate: plant sphingosine: mannose = 0.1: 0.01: 0.1. the weight ratio of the second additive formula is H2O2：K2NO(SO3)2=0.01：0.5。
) Harvest of the fermentation
Continuously culturing the fermentation product for 20 days at 17 ℃ under the conditions of ventilation volume of 1:1 (v/v), pressure of 0.1MP, heating to 50.0 ℃ and keeping for 5 minutes; cooling, adding ethanol with a volume 0.1 times of the volume of the fermented product, and standing at 0 ℃ for 1 hour; then centrifuging for 5 minutes at 5000 rpm, or sieving by a 50-mesh sieve and filtering; concentrating the supernatant or filtrate to 1/10, purifying with reverse phase carbon 18 chromatographic column with mobile phase of 0% ethanol, collecting eluate after sample injection for 2 min, and removing solvent under reduced pressure of 0.0001Mpa to obtain the immunopotentiator. The component analysis shows that the immunopotentiator contains 5% of saccharides, 3% of amino acids and derivatives thereof, and 1% of nucleosides. While obtaining the immunopotentiator, the hypha obtained by centrifugal precipitation or filtration can also replace cordyceps sinensis to process related health products.
Example 2
A preparation operation step of an immunopotentiator based on hirsutella sinensis comprises the following steps:
1) strain selection
Cordyceps collected from Qinghai [ (C.) A. Ex Fr. ] SingOphiocordyceps sinensisHirsutella sinensis isolated from stroma (Hirsutella sinensis）；
2) Culturing of bacterial strains
The culture method is liquid-solid mixed culture and comprises the following specific steps:
2.1) first order solid Strain preparation
Inoculating the separated and purified strains into a solid culture medium of a 9cm culture dish, wherein the culture medium comprises: 200.0 g of potato cooking juice, 20.0 g of glucose, 10.0 g of maltose, 10.0 g of peptone, 10.0 g of yeast powder and MgSO4 1.5 g、KH2PO43.0 g of agar, 20.0 g of agar, 1.0L of water, and adjusting the pH to 6.5 by using 1.0M NaOH; placing into an incubator at 20 deg.C for 30 days to obtain first-stage solid strain.
) Second-stage liquid shake flask seed culture
200mL of liquid culture medium is filled into a 500mL triangular flask, and the culture medium contains 20.0 g of glucose, 30.0 g of silkworm chrysalis powder, 20.0 g of yeast powder, 40.5 g of MgSO40.5g and KH in 1000mL2PO41.0 g, mixing the components, adding water to supplement the mixture to 1000mL, and adjusting the pH value to 6.5 by using 1.0M NaOH to obtain a liquid culture medium; using 1.2 Kg/cm2Autoclaving for 30 min, cooling to room temperature, inoculating 2 solid strains in culture dish to 500mL triangular flask culture medium, and culturing at 20 deg.C and 200 rpm in a constant temperature shaking incubator for 20 days to obtain second-stage liquid strain.
) Preparing three-stage liquid strain
Inoculating the secondary liquid strain into a seed tank of a fermentation system according to the volume ratio of 5%, wherein the liquid filling amount of a liquid culture medium of the fermentation tank is 70%; culturing for 5 days at 20 deg.C under the conditions of ventilation volume of 1:1 (v/v) and pressure of 0.1MP to obtain three-stage liquid strain;
the formula of the liquid culture medium is the same as that of the liquid culture medium used by the secondary liquid strain.
) Quaternary fermentation and metabolic regulation
Inoculating the third-stage liquid strain into a fermentation tank of a fermentation system according to the volume ratio of 10%, wherein the liquid loading of a fermentation culture medium is 70%, and each liter of the fermentation culture medium contains 20g of glucose, 30g of silkworm chrysalis meal,Yeast powder 20g, MgSO4 0.5g、KH2PO41g, pH 6.5. Adding 1000mg/L of additive I into the culture medium for 30 days at 20 ℃ and the aeration rate of 1:1 (v/v) by volume and the pressure of 0.1 MP; continuing culturing for 40 days, adding 100.0 mg/L second additive, and continuing culturing for 40 days to obtain fermented product;
the formula of the first additive comprises zinc sulfate: plant sphingosine: mannose = 0.9: 0.2: 0.5. the weight ratio of the second additive formula is H2O2：K2NO(SO3)2=0.8：0.01。
) Harvest of the fermentation
Heating the fermentation to 99.9 deg.C for 20 min; cooling, adding ethanol with the volume 2.0 times of the volume of the fermentation product, and standing at 20 ℃ for 24 hours; then centrifuging for 15 minutes at 10000 rpm or filtering by a 200-mesh sieve; concentrating the supernatant or filtrate to 1/2, purifying with reverse phase carbon 18 chromatographic column with mobile phase of 90% ethanol, collecting eluate after sample injection for 50 min, and removing solvent under 0.01MPa to obtain the immunopotentiator. The component analysis shows that the immunopotentiator contains 50% of saccharides, 30% of amino acids and derivatives thereof, and 10% of nucleosides. While obtaining the immunopotentiator, the hypha obtained by centrifugal precipitation or filtration can also replace cordyceps sinensis to process related health products.
Example 3
A preparation operation step of an immunopotentiator based on hirsutella sinensis comprises the following steps:
1) strain selection
Cordyceps collected from Qinghai [ (C.) A. Ex Fr. ] SingOphiocordyceps sinensisHirsutella sinensis isolated from stroma (Hirsutella sinensis）；
2) Culturing of bacterial strains
The culture method is liquid-solid mixed culture and comprises the following specific steps:
2.1) first order solid Strain preparation
The separated and purified strain was inoculated into a solid medium (culture medium: potato 200.0 g broth, glucose 20.0 g, maltose 10.0 g, peptone 10.0 g, yeast powder 10) in a 9cm petri dish.0 g、MgSO4 1.5 g、KH2PO43.0 g of agar, 20.0 g of agar, 1.0L of water, adjusting the pH to 6.5 with 1.0M NaOH), and placing in an incubator at 18.5 ℃ for 25 days to obtain a first-stage solid strain.
) Second-stage liquid shake flask seed culture
200mL of liquid culture medium is filled into a 500mL triangular flask, and the culture medium contains 20.0 g of glucose, 30.0 g of silkworm chrysalis powder, 20.0 g of yeast powder, 40.5 g of MgSO40.5g and KH in 1000mL2PO41.0 g, mixing the components, adding water to supplement the mixture to 1000mL, and adjusting the pH value to 6.5 by using 1.0M NaOH to obtain a liquid culture medium; using 1.2 Kg/cm2Autoclaving for 30 min, cooling to room temperature, inoculating 2 solid strains in culture dish to 500mL triangular flask culture medium, and culturing in constant temperature shaking incubator at 18.5 deg.C and 180 rpm for 15 days to obtain second-stage liquid strain.
) Preparing three-stage liquid strain
Inoculating the secondary liquid strain into a seed tank of a fermentation system according to the volume ratio of 5%, wherein the liquid filling amount of a liquid culture medium of the fermentation tank is 70%; the ventilation volume is 1:1 (v/v) by volume, and the pressure is 0.1 MP; culturing at 20 deg.C for 15 days to obtain third-stage liquid strain;
the formula of the liquid culture medium is the same as that of the liquid culture medium used by the secondary liquid strain.
2.4) Quaternary fermentation and Metabolic Regulation
Inoculating the third-stage liquid strain into a fermentation tank of a fermentation system according to the volume ratio of 10%, wherein the liquid loading amount of a fermentation culture medium is 60%, and each liter of the fermentation culture medium contains 20g of glucose, 30g of silkworm chrysalis powder, 20g of yeast powder and MgSO4 0.5g、KH2PO41g, pH 6.5. Adding 500mg/L additive into the culture medium for 30 days at 20 ℃ and the aeration rate of 1:1 (v/v) by volume and 0.1MP by pressure; continuously culturing for 40 days, and adding 50.0 mg/L second additive agent; further culturing for 30 days to obtain fermented product.
The formula of the first additive comprises zinc sulfate: plant sphingosine: mannose = 0.5: 0.1: 0.3. the weight ratio of the second additive formula is H2O2：K2NO(SO3)2=0.4：0.2。
) Harvest of the fermentation
Heating the fermentation to 80.0 ℃ for 10 minutes; cooling, adding ethanol with volume 1.0 times of the volume of the fermented product, and standing at 10 deg.C for 12 hr; centrifuging at 7500 rpm for 10 min, or sieving with 100 mesh sieve; concentrating the supernatant or filtrate to 1/5, purifying with reversed phase carbon 18 chromatographic column with mobile phase of 45% ethanol, collecting eluate 30 min after sample injection, and removing solvent at 0.001Mpa to obtain the immunopotentiator. The component analysis shows that the immunopotentiator contains 30% of saccharides, 15% of amino acids and derivatives thereof, and 5% of nucleosides. While obtaining the immunopotentiator, the hypha obtained by centrifugal precipitation or filtration can also replace cordyceps sinensis to process related health products.
Claims (8)
1. An immunopotentiator based on hirsutella sinensis is characterized in that: the immunopotentiator is white dry powder; wherein the content of saccharides is 5-50%, the content of amino acids and derivatives thereof is 3-30%, and the content of nucleosides is 1-10%;
the immunopotentiator is prepared by the following steps:
(1) preparation of first-order solid Strain
The separated and purified hirsutella sinensis (A), (B), (C), (Hirsutella sinensis) Inoculating slant strains in a flat solid culture medium, wherein the inoculation amount is that each slant is inoculated with 2 plates of 9 cm; culturing at 17-20 ℃ for 20-30 days to obtain a first-grade solid strain;
(2) preparation of second-stage liquid strain
Inoculating the first-stage solid strain in a liquid culture medium, wherein the inoculation amount is that 1 plate of the first-stage solid strain is inoculated in every 100mL of the liquid culture medium; shaking culture: the temperature is 17-20 ℃, and the rotating speed is 160-200 r/min; culturing for 10-20 days to obtain a secondary liquid strain;
(3) preparing three-stage liquid strain
Inoculating the secondary liquid strain into a seed tank of a fermentation system according to the volume ratio of 5-20%, wherein the liquid filling amount of a liquid culture medium of the fermentation tank is 50-70%; culturing for 5-15 days at 17-20 ℃ according to the volume ratio of 1:1 (v/v) and the pressure of 0.1MP to obtain a third-level liquid strain;
the formula of the liquid culture medium is the same as that of the liquid culture medium used by the secondary liquid strain;
(4) preparation of fermentation broth
Inoculating the third-stage liquid strain into a fermentation tank of a fermentation system according to the volume ratio of 5-10%, wherein the liquid filling amount of the fermentation tank is 50-70%; the ventilation volume is 1:1 (v/v) by volume, the pressure is 0.1MP, and the first additive with the volume ratio of 100-1000 mg/L is added after the culture is carried out for 5-30 days at the temperature of 17-20 ℃; continuously culturing for 10-40 days, adding 1.0-100.0 mg/L second additive, and continuously culturing for 20-40 days; obtaining fermentation liquor;
the fermentation medium and the liquid medium have the same formula;
the additive No. one: is prepared from zinc sulfate: plant sphingosine: mannose according to a weight ratio of 0.1-0.9: 0.01-0.2: 0.1-0.5;
the second additive: hydrogen peroxide (H)2O2): potassium nitrosodisulfonate (K)2NO(SO3)2) According to the weight ratio of 0.01-0.8: 0.5-0.01;
(5) preparation of hirsutella sinensis immunopotentiator
Heating the fermentation liquor to 50.0-99.9 ℃, and keeping for 5-20 minutes; cooling to room temperature, adding ethanol with the volume of 0.1-2.0 times of that of the fermentation liquor, standing for 1-24 hours at the temperature of 0-20 ℃; centrifuging to obtain supernatant or filtering to obtain filtrate; concentrating the supernatant or the filtrate to 1/10-1/2 of the original volume, passing through a reversed phase carbon 18 chromatographic column, eluting with 0-90% ethanol, collecting the eluate after sample injection for 2-50 minutes, removing the solvent in the eluate, and recovering the ethanol to obtain the hirsutella sinensis immunopotentiator.
2. The immunopotentiator according to claim 1, wherein: in the preparation step (1), the solid medium: 200g of potato cooking juice, 20g of glucose, 10g of maltose, 10g of peptone, 10g of yeast powder and magnesium sulfate (MgSO 2)4) 1.5g, potassium dihydrogen phosphate (KH 2 PO)4) 3g of agar, 20g of agar, 1L of water, 1M sodium hydroxide(NaOH) to pH 6.5.
3. The immunopotentiator according to claim 1, wherein: in the preparation steps (2) and (3), the liquid medium: 20g of glucose, 30g of silkworm chrysalis powder, 20g of yeast powder and magnesium sulfate (MgSO)4) 0.5g of potassium dihydrogen phosphate (KH 2 PO)4) 1g of water was added to 1L, and the pH was adjusted to 6.5 with 1M sodium hydroxide (NaOH).
4. The immunopotentiator according to claim 1, wherein: in the preparation step (4), the fermentation medium: 20g of glucose, 30g of silkworm chrysalis powder, 20g of yeast powder and magnesium sulfate (MgSO)4) 0.5g of potassium dihydrogen phosphate (KH 2 PO)4) 1g of water was added to 1L, and the pH was adjusted to 6.5 with 1M sodium hydroxide (NaOH).
5. The immunopotentiator according to claim 1, wherein: in the preparation step (5), the supernatant or the filtrate is concentrated to 1/10-1/2 of the original volume under the pressure of 0.0001-0.01 Mpa under reduced pressure.
6. The immunopotentiator according to claim 1, wherein: in the preparation step (5), the centrifugation: rotating speed of 5000-10000 r/min, and separating for 5-15 min.
7. The immunopotentiator according to claim 1, wherein: in the preparation step (5), the filtration: sieving the mixture by a sieve of 50-200 meshes.
8. The immunopotentiator according to claim 1, wherein: in the preparation step (5), the mobile phase passing through the reversed phase carbon 18 chromatographic column is 0-90% ethanol.
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